I have an Arabidopsis protein, which should be apoplastic (rice homologue is localized to apoplast and this protein contains predicted signal peptide).
I expressed the protein without the signal peptide (but I have also version with the SP ready) with the alpha factor under control of AOX1 promotor. After dilution of O/N preculture, I expressed the yeast (also plasmid control, which should express protein in frame with the C-term tags) in the BMXY medium, which I supplemented with half of glycerol and half with methanol. Next 4 days, I supplemented methanol to keep the induction and I collected samples every 24 hrs, when I centrifuged 1 ml of medium and froze the supernatant and pellet (cells) separately.
As I did not detect activity in the supernatant previously ( https://www.researchgate.net/post/Can_a_plant_cell_wall-associated_protein_remain_in_the_cell_wall_when_heterologously_expressed_in_yeast_Pichia_pastoris ), I extracted proteins from the cells and from cell wall and loaded them on SDS-PAGE. I wanted to load the same amount of proteins to each well, so I loaded rather low amount, because medium contained only low concentration.
I have used an old polyclonal anti c-Myc antibody, but there was nothing on the blot, except of some non-specific bands in the control.
As there is no clear band neither on SDS-PAGE, nor on Western blot, I would consider this as negative results. So what can I try next?
First, I could try gel and blot with more proteins. However, since there is no obvious large band, does it make sense?
Second, I could check all timepoints for expressed protein.
Third, mRNA analysis.
What should I do next? What makes sense and what doesn't?
Thank you
Hi Tomas,
Well, I feel your pain, since we often had this difficulty (primarily with rice proteins, so a bit similar). You did not mention was this zeocin selection or what selection and how many colonies did you screen? Usually, we screened 20-100 colonies to find the highest expresser and many did not express much. However, even then, we sometimes got nothing obvious and eventually gave up. Looking at the RNA expression is probably a good idea, although we have not done that. If you have not confirmed the gene insertion, you could try colony PCR or other DNA techniques to at least confirm the gene. You could consider codon usage and such, but I guess you may have done that already.
Good luck!
Hello James Ketudat Cairns
thank you for your answer.
Sorry for the lack of information. The plasmid is pPICZalpha, so with Zeo selection. I did not use it during the expression, though (I found on Friday this https://www.researchgate.net/post/Why_does_Pichia_pastoris_expression_seem_to_fail_when_grown_without_Zeocin - I can try to add it next time, but I would not expect to save they day completely).
I did confirm insertion of my gene into DNA by PCR.
Well, I tried only 2 clones, because I have 4 versions of my protein and I do not have exactly established way to determine my protein. I have not (successfully) measured activity (yet). For Western blot I have polyclonal antibodies, which bind something else in the yeast extract (so dot blot is not an option) and SDS-PAGE is obviously not very infromative.
Another problem is, that I am not quite sure, where to look for my protein :(
https://www.researchgate.net/post/Can_a_plant_cell_wall-associated_protein_remain_in_the_cell_wall_when_heterologously_expressed_in_yeast_Pichia_pastoris
What was your protocol for trying 20+ clones? (in what volume did you grow the yeast, for how long, how did you detect your protein?)
Thank you very much, I appreciate any help!
Normally, we screen by expression in 5 mL cultures (in 50 mL conical tubes) according to the Pichia Manual (although we also use pPICZalpha derivatives which came out after the original manual). We screen by enzyme assays (synthetic colorimetric substrates), but also by SDS-PAGE. Although I tend to discourage using zeocin when expressing, since I have to pay the bills, the students may add it at this step. We tried tagging with eGFP to see if proteins were retained in the vacuole before, but it did not seem particularly effective, probably because expression was quite low.
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